Three-dimensional structure of manganese superoxide dismutase from Bacillus halodenitrificans,a component of the so-called "green protein"

A so-called "green protein" has been purified from a moderate halophilic eubacterium, Bacillus halodenitrificans (ATCC 49067), under anaerobic conditions. The protein, which might play an important role in denitrification, dissociates mainly into two components after exposure to air: a manganese superoxide dismutase (GP-MnSOD) and a nucleoside diphosphate kinase. As a first step in elucidating the overall structure of the green protein and the role of each component, the 2.8-A resolution crystal structure of GP-MnSOD was determined. Compared with other manganese dismutases, GP-MnSOD shows two significant characteristics. The first is that the entrance to its substrate channel has an additional basic residue-Lys38. The second is that its surface is decorated with an excess of acidic over basic residues. All these structural features may be related to GP-MnSOD's high catalytic activity and its endurance against the special cytoplasm of B. halodenitrificans. The structure of GP-MnSOD provides the basis for recognizing its possible role and assembly state in the green protein.     

Bovine floor plate explants secrete SCO-spondin

SCO-spondin is a large-molecular mass glycoprotein, secreted by the subcommissural organ (SCO), which has been implicated in neuronal development during ontogeny of the central nervous system. The expression of SCO-spondin is not restricted to the SCO but it also occurs in the floor plate, a key structure participating in neuronal differentiation and patterning of the neural tube. It has been postulated that SCO-spondin detected in the floor plate is released into the lumen of the neural tube, but this new route of secretion of floor plate cells needs to be further substantiated. For this purpose, we standardized long-term organ culture of bovine floor plate and performed morphological, immunological, biochemical, and gene expression analyses. The study of floor plate explants and their conditioned media allowed us to demonstrate that: (1) organ-cultured floor plate cells are actively secretory for up to 25 days; (2) SCO-spondin gene is actively transcribed and translated by the cultured floor plate cells; (3) SCO-spondin is released into the culture medium via the apical cell pole; and (4) upon release, SCO-spondin does not aggregate in the conditioned medium but remains soluble. Furthermore, in the cultured floor plate cells, SCO-spondin may be secreted through a route bypassing the Golgi apparatus.     

Preliminary crystallographic studies of two C-terminally truncated copper-containing nitrite reductases from Achromobacter cycloclastes: changed crystallizing behaviors caused by residue deletion

The C-terminal segment of copper-containing nitrite reductase from Achromobacter cycloclastes (AcNiR) has been found essential for maintaining both the quaternary structure and the enzyme activity of AcNiR. C-terminal despentapeptide AcNiR (NiRc-5) and desundecapeptide AcNiR (NiRc-11) are two important truncated mutants whose activities and stability have been affected by residue deletion. In this study, the two mutants were crystallized using the hanging drop vapor diffusion method. Crystals of NiRc-5 obtained at pH 5.0 and 6.2 both belonged to the P2(1)2(1)2(1) space group with unit cell parameters a=99.0 A, b=117.4 A, c=122.8 A (pH 5.0) and a=98.9A, b=117.7A, c=123.0A (pH 6.2). NiRc-11 was crystallized in two crystal forms: the tetragonal form belonged to the space group P4(1) with a=b=96.0A and c=146.6A; the monoclinic form belonged to the space group P2(1) with a=86.0A, b=110.1A, c=122.7A, and beta=101.9 degrees. The crystallizing behaviors of the two mutants differed from that of the native enzyme. Such change in combination with residue deletion is also discussed here.     

Regulation of ion channels by protein tyrosine phosphorylation

Ion channels are regulated by protein phosphorylation and dephosphorylation of serine, threonine, and tyrosine residues. Evidence for the latter process, tyrosine phosphorylation, has increased substantially since this topic was last reviewed. In this review, we present a comprehensive summary and synthesis of the literature regarding the mechanism and function of ion channel regulation by protein tyrosine kinases and phosphatases. Coverage includes the majority of voltage-gated, ligand-gated, and second messenger-gated channels as well as several types of channels that have not yet been cloned, including store-operated Ca2+ channels, nonselective cation channels, and epithelial Na+ and Cl- channels. Additionally, we discuss the critical roles that channel-associated scaffolding proteins may play in localizing protein tyrosine kinases and phosphatases to the vicinity of ion channels.     

Interleukin-1beta, Src- and non-Src tyrosine kinases, and nitric oxide synthase induction in rat aorta in vitro

We studied the potential roles for endogenous interleukin-1beta (IL-1beta) and for several signaling pathways in the spontaneous induction in vitro of inducible nitric oxide synthase (iNOS) in endothelium-denuded rat aorta rings. Added IL-1beta augmented, whereas the IL-1beta receptor antagonist IL-1ra blocked, spontaneous iNOS induction. Furthermore, increases in IL-1beta mRNA preceded those of iNOS mRNA. Mitogen-activated protein kinase kinase and phosphatidyl inositol 3' kinase inhibition did not block iNOS induction, whereas nuclear factor kappaB inhibition did. The sarcoma virus tyrosine kinase (Src) family-selective inhibitor 4-amino-5(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) blocked the upregulation of IL-1beta mRNA and the subsequent induction of iNOS but not the induction of iNOS stimulated by exogenously added IL-1beta. In contrast, the non-Src inhibitors TP 47/AG 213 and genistein and the tyrosine phosphatase inhibitor vanadate did not affect the spontaneous upregulation of IL-1beta mRNA but blocked both the IL-1beta-mediated and spontaneous induction of iNOS. We conclude that 1) the upregulation of tissue IL-1beta, via a signaling pathway involving a Src family kinase, plays a key role in rat vascular iNOS induction and 2) non-Src tyrosine kinases play roles downstream from IL-1beta for iNOS induction.     

银鲫复合种外源遗传物质整入的RAPD分析

在优化ＲＡＤＰＤ检测条件的基础上,采用４０对随机引物,比较分析了银复合物,异育银鲫和兴国红鲤相互间扩增ＤＮＡ片段的异同性。总的来说,银鲫复合种和异育银鲫具有基本一致的扩增产物,而与兴国红鲤的扩增产物多数不同,相似率分析表明,银鲫复合种与兴国红鲤之间的相似率为３１．６％,异育银鲫与兴国红鲤之间的相似率为２８．６％。在分析中,除发现银鲫复合种,异育银鲫与兴国红鲤间共有扩增片段外,还发现了银鲫复合种与兴     

人血小板生成素氨基端功能区cDNA的克隆及其定点突变

以Nested－PCR方法从人肝cDNA基因文库中扩增出编码人血小板生成素（hTPO）前153个氨基酸的氨基端功能区cDNA;在扩增中,采用非连续多核甘酸定点突变的方法．将翻译起始的七个氨基酸的原核中不常用的密码子同又突变成使用频率较高的密码子,以便于其在大肠杆菌中表达。序列测定证实了预期的结果。     

维甲酸对鼻咽癌细胞生长、表型和瘤基因表达的作用

研究了维甲酸（ＲＡ）对鼻咽癌细胞生长、表型和癌基因表达的作用．用ＲＡ诱导鼻咽癌细胞,绘制诱导前后的细胞曲线,观察细胞形态,并用Ｎｏｒｔｈｅｒｎ杂交和ＤＮａｓｅⅠ超敏感区分析法检测基因表达和调控．结果表明,ＲＡ能显著抑制鼻咽癌细胞的生长,前５ｄ下降约５０％．ＲＡ处理后的细胞从典型的多边形形态变成扁平、细长,类似纤维细胞状的形态．ＲＡ诱导前ｃ－ｍｙｃ基因和ｃ－Ｈａ－ｒａｓ基因ＨＮＥ２细胞中高表达,而诱导后ｃ－ｍｙｃ基因表达水平急剧下降,ｃ－Ｈａ－ｒａｓ基因无明显改变．在实验中还发现ＲＡ诱导前后的ｃ－ｍｙｃ基因和ｃ－Ｈａ－ｒａｓ基因中一些重要的超敏感位点和它们的功能．由实验结果可得到如下结论：ＲＡ能促进鼻咽癌细胞分化,通过对染色体上调控位点的作用来抑制ｃ－ｍｙｃ基因的表达,ＤＮａｓｅⅠ超敏感位点与细胞的分化程度、细胞的组织特异性和基因表达状态有关,ｃ－ｍｙｃ基因可通过不同的调控方式而失活．